Methods for illuminating a specimen are used in the field, in particular in confocal microscopy. In this context, a specimen is scanned by a mostly laser-produced light beam in a line-by-line or meander pattern. However, in order to avoid damaging the specimen, only specific specimen regions, the regions of interest (ROIs), are illuminated. Since the light beam, conditionally upon the inertia of the light beam deflection, sweeps over the entire specimen, its intensity is modulated as a function of the position on the specimen. The information indicating whether a point of a specimen is to be illuminated is stored in a memory and is read out from the same on the basis of the light beam position. The control data are then fed to the light modulator.
If the illuminated specimen is made up of cells in an aqueous solution, for example, then the position, orientation, and shape of the specimen or of the ROI do not remain constant. The changes in the specimen are mapped by adapting the intensity data in the memory. In the process, however, it may be necessary to update large areas of the memory. However, since the process of updating the memory contents and reading out from the same must take place in parallel and in real time, rigorous demands are placed on the speed of the memory used.
This becomes especially problematic when specimens are to be scanned at a high frame rate. In such a case, the available memories quickly reach their performance limits and become very costly. In addition, very high-performance processors are needed for computing the new memory contents.
In a schematic representation, FIG. 1 shows the logical sequence of an illuminating method. In this case, the deflecting electronics of laser beam 1, which controls the position of the laser beam on the specimen for scanning the same in a line-by-line pattern, generates a value for x-position 2 and for y-position 3 of the laser beam. These two values are supplied to an address generator 4, which, in turn, from these values, generates a memory address 5 in memory 6. Intensity data 7, which are used to influence the intensity of the light beam via interface 8, driver 9, and crystal 10, are stored in memory 6. Interface 8, driver 9 and crystal 10 may be AOTF components. In this method, a change in the specimen or in a region of the specimen is mapped in an adaptation 11 of the contents of memory 6.